Mechanisms of assembly of photoreceptor G protein complexes

 描述（由申请人提供）：正确的蛋白质折叠是维持健康神经元（包括视网膜感光细胞）的关键问题。光感受器蛋白质组的应激可能是由环境因素如光损伤或遗传因素如视紫红质和许多其他光感受器蛋白质的错误折叠突变引起的。蛋白质组由一类称为分子伴侣的蛋白质维持。这些分子伴侣保护蛋白质免于聚集，引导其折叠途径并促进其结合成多蛋白质组装体。一种重要类型的分子伴侣是在真核细胞质溶胶中发现的II型伴侣蛋白，称为CCT（含有无尾复合物多肽1的细胞质伴侣蛋白，也称为TRiC）。已知需要CCT进行折叠的蛋白质数量有数百种，并且还在继续增长。其中包括G蛋白β亚基（Gβ），其形成Gβγ和Gβ5-RGS（G蛋白信号调节剂）二聚体，这些二聚体是视觉信号的关键组分。Gβγ和RGS-Gβ5也需要CCT共伴侣蛋白，即糖蛋白样蛋白，以进行适当的折叠和二聚体形成。在以前的工作中，我们已经确定了两个中间体的结构，在Gβγ组装低温电子显微镜，化学交联与质谱联用，并与非天然氨基酸的位点特异性交联。这些结构为Gβγ组装机制提供了分子细节。在目标1中，我们建议确定RGS-Gβ5组装中类似中间体的结构，以在分子水平上了解CCT和PhLP 1如何帮助RGS-Gβ5二聚体形成。 CCT在Bardet-Biedl综合征（BBS）中的额外作用也已得到证实。BBS是一种遗传性睫状体功能障碍性疾病，其特征在于包括视网膜变性在内的多种病理状况。BBS是由不能形成BBSome引起的，BBSome是八种蛋白质的复合物，对于囊泡运输到纤毛是必需的。CCT和CCT样复合物由三种蛋白质组成，已知其突变导致BBS（BBS 6，10和12）是BBSome组装所需的。在目标2中，我们提出了类似的结构研究，以确定BBSome组装和BBSome功能的分子机制。 从这些研究中获得的信息对于设计基于伴侣的方法来治疗由RGS-Gβ5和BBSome功能障碍引起的视网膜疾病至关重要。

 DESCRIPTION (provided by applicant): Proper protein folding is a key issue in maintaining healthy neurons, including the photoreceptor cells of the retina. Stresses on the photoreceptor proteome may be caused by environmental factors such as light damage or by genetic factors such as misfolding mutations in rhodopsin and many other photoreceptor proteins. The proteome is maintained by a class of proteins called molecular chaperones. These chaperones protect proteins from aggregation, channel their folding pathways and facilitate their association into multi-protein assemblies. An important type of molecular chaperone is the type II chaperonin found in the eukaryotic cytosol, termed CCT (Cytosolic Chaperonin containing Tailless complex polypeptide 1, also called TRiC). The number of proteins known to require CCT for their folding is in the hundreds and continues to grow. Among these are the G protein β subunits (Gβ) which form the Gβγ and Gβ5-RGS (Regulator of G protein Signaling) dimers that are key components in visual signaling. Both Gβγ and RGS-Gβ5 also require the CCT co-chaperone, phosducin-like protein, for proper folding and dimer formation. In previous work, we have determined the structures of two intermediates in Gβγ assembly by cryo-electron microscopy, chemical cross-linking coupled with mass spectrometry, and site-specific cross-linking with unnatural amino acids. These structures have provided molecular detail into the mechanism of Gβγ assembly. In Aim 1, we propose to determine the structures of similar intermediates in RGS-Gβ5 assembly to understand at the molecular level how CCT and PhLP1 assist in RGS-Gβ5 dimer formation. An additional role for CCT in Bardet-Biedl syndrome (BBS) has also been demonstrated. BBS is a genetic disease of ciliary dysfunction characterized by multiple pathological conditions including retinal degeneration. BBS is caused by an inability to form the BBSome, a complex of eight proteins that is essential for vesicle trafficking to cilia. CCT and a CCT-like complex made up of three proteins whose mutations are known to cause BBS (BBS 6, 10 and 12) are required for the assembly of the BBSome. In Aim 2, we propose similar structural studies to determine the molecular mechanism of BBSome assembly and BBSome function. The information gained from these studies will be vital in designing chaperone-based methods to treat retinal diseases caused by RGS-Gβ5 and BBSome malfunctions.